Abstract

Concerted Applications of Recombineering and CRISPR/Cas9 Assisted Targeting

Francis Stewart, Professor, Technische Universitat Dresden

Two concerted applications of recombineering and Cas9-assisted targeting will be described. A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple a new recombineering application for the rapid generation of short-arm targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. Amongst several applications, we focus on biallelic protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Despite high frequencies of monoallelic targeting, we found that simultaneous biallelic targeting requires long-arm targeting constructs. Second, Cas9 assisted targeting has been applied for large genome engineering to humanise mouse genes from start to stop codons. Hybrid targeting constructs using mouse homology arms either side of the corresponding human gene were introduced into ESCs by Cas9 assisted targeting at good frequencies.